Many members of the tumor necrosis factor (TNF) family of ligands and their corresponding receptors regulate growth of normal cells by inducing apoptosis or enhancing cell survival and proliferation. It is this balance between apoptotic signals and survival and proliferation signals that maintains normal cellular homeostasis. At least 26 TNF family receptors and 18 TNF family ligands have been identified to date. The biologically active forms of both the receptors and ligands are self-assembled protein trimers. Transmembrane and soluble forms of both the receptors and ligands have been identified. Though the intracellular domains of the receptors share no sequence homology, their extracellular domains comprise 40-amino-acid, cysteine-rich repeats. Their cytoplasmic tails signal by interacting with two major groups of intracellular proteins: TNF receptor-associated factors (TRAFs) and death domain (DD)-containing proteins. Interaction between at least six human TRAFs and TRAF-binding sites on the cytoplasmic tail of some of these receptors initiates several signaling pathways, including AKT (the serine/threonine kinase referred to as protein kinase B or PKB), nuclear factor-κb (NF-κB), and mitogen-activated protein kinases (MAPK). See, for example, the review by Younes and Kadin (2003) J. Clin. Oncol. 18:3526-3534.
The TNF family receptor member CD40 is a 50-55 kDa cell-surface antigen present on the surface of both normal and neoplastic human B cells, dendritic cells, monocytes, macrophages, CD8+ T cells, endothelial cells, monocytic and epithelial cells, some epithelial carcinomas, and many solid tumors, including lung, breast, ovary, urinary bladder, and colon cancers. Binding of the CD40 ligand to the CD40 antigen on the B cell membrane provides a positive costimulatory signal that stimulates B cell activation and proliferation, resulting in B cell maturation into a plasma cell that secretes high levels of soluble immunoglobulin. CD40 activates TRAF-1, TRAF-2, -3, -5, and -6, which upregulate diverse signaling pathways following engagement of CD40 with CD40L (either membrane-bound CD40L or soluble CD40L), including extracellular signal-regulated kinase (ERK), c-jun amino terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), AKT, and NF-κB (see, for example, Younes and Carbone (1999) Int. J. Biol. Markers 14:135-143; van Kooten and Banchereau (2000) J. Leukoc. Biol. 67:2-17).
Malignant B cells from tumor types of B-cell lineage express CD40 and appear to depend on CD40 signaling for survival and proliferation. Transformed cells from patients with low- and high-grade B-cell lymphomas, B-cell acute lymphoblastic leukemia, multiple myeloma, chronic lymphocytic leukemia, Walsdenstrom's Macroglobulinemia, and Hodgkin's disease express CD40. CD40 expression is also detected in two-thirds of acute myeloblastic leukemia cases and 50% of AIDS-related lymphomas.
A number of carcinomas and sarcomas also exhibit high levels of CD40 expression, though the role of CD40 signaling in relation to CD40 expression on these cancer cells is less well understood. CD40-expressing carcinomas include urinary bladder carcinoma (Paulie et al. (1989) J. Immunol. 142:590-595; Braesch-Andersen et al. (1989) J. Immunol. 142:562-567), breast carcinoma (Hirano et al. (1999) Blood 93:2999-3007; Wingett et al. (1998) Breast Cancer Res. Treat. 50:27-36); prostate cancer (Rokhlin et al. (1997) Cancer Res. 57:1758-1768), renal cell carcinoma (Kluth et al. (1997) Cancer Res. 57:891-899), undifferentiated nasopharyngeal carcinoma (UNPC) (Agathanggelou et al. (1995) Am. J. Pathol. 147:1152-1160), squamous cell carcinoma (SCC) (Amo et al. (2000) Eur. J. Dermatol. 10:438-442; Posner et al. (1999) Clin. Cancer Res. 5:2261-2270), thyroid papillary carcinoma (Smith et al. (1999) Thyroid 9:749-755), cutaneous malignant melanoma (van den Oord et al. (1996) Am. J. Pathol. 149:1953-1961), gastric carcinoma (Yamaguchi et al. (2003) Int. J. Oncol. 23(6):1697-702), and liver carcinoma (see, for example, Sugimoto et al. (1999) Hepatology 30(4):920-26, discussing human hepatocellular carcinoma). For CD40-expressing sarcomas, see, for example, Lollini et al. (1998) Clin. Cancer Res. 4(8):1843-849, discussing human osteosarcoma and Ewing's sarcoma.
CD40 signaling protects immature B-cells and B-cell lymphomas from apoptosis induced by IgM or Fas (see, for example, Wang et al. (1995) J. Immunol. 155:3722-3725). Mantel cell lymphoma cells express a high level of CD40, and the addition of exogenous CD40 ligand was shown to enhance their survival and rescue them from fludarabin-induced apoptosis (Clodi et al. (1998) Brit. J. Haematol. 103:217-219).
The role of CD40 signaling in malignant B cell survival and proliferation renders the CD40 antigen a potential target for anti-cancer therapy. Indeed, antagonist anti-CD40 antibodies inhibit proliferation and/or differentiation of malignant human B cells in vitro (see, for example, U.S. Patent Application Publication No. 20040109857). Further, murine models of aggressive human lymphomas have demonstrated the in vivo efficacy of anti-CD40 antibodies in promoting animal survival. See, for example, Funakoshi et al. (1994) Blood 83:2787-2794; Tutt et al. (1998) J. Immunol. 161:3176-3185; and Szocinski et al. (2002) Blood 100: 217-223.
The CD40 ligand (CD40L), also known as CD154, is a 32-33 kDa transmembrane protein that also exists in two smaller biologically active soluble forms, 18 kDa and 31 kDa, respectively (Graf et al. (1995) Eur. J. Immunol. 25:1749-1754; Mazzei et al. (1995) J. Biol. Chem. 270:7025-7028; Pietravalle et al. (1996) J. Biol. Chem. 271:5965-5967). CD40L is expressed on activated, but not resting, CD4+ T-helper cells (Lane et al. (1992) Eur. J. Immunol. 22:2573-2578; Spriggs et al. (1992) J. Exp. Med. 176:1543-1550; and Roy et al. (1993) J. Immunol. 151:1-14). Both CD40 and CD40L have been cloned and characterized (Stamenkovi et al. (1989) EMBO J. 8:1403-1410; Armitage et al. (1992) Nature 357:80-82; Lederman et al. (1992) J. Exp. Med. 175:1091-1101; and Hollenbaugh et al. (1992) EMBO J. 11:4313-4321). See also U.S. Pat. No. 5,945,513, describing human CD40L. Cells transfected with the CD40L gene and expressing the CD40L protein on their surface can trigger B-cell proliferation, and together with other stimulatory signals, can induce antibody production (Armitage et al. (1992) supra; and U.S. Pat. No. 5,945,513). Patients with lymphoid malignancies, autoimmune disease, cardiovascular disease, and essential thrombocythemia have elevated serum levels of soluble CD40L (sCD40L) that are not seen in healthy subjects. Constitutive expression of CD40L has been observed in a subset of patients with several B-cell lymphoid malignancies, including mantle-cell lymphoma, follicular lymphoma, marginal zone lymphoma, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and HIV-infected B-cell lymphoma. See, for example, Clodi et al. (1998) Br. J. Haematol. 103:270-275; Schattner et al. (1998) Blood 91:2689-2697; Moses et al. (1997) Nat. Med. 3:1242-1249; Trentin et al. (1997) Cancer Res. 57:4940-4947; and Pham et al. (2002) Immunity 16:37-50). CD40L may play an important role in the cell contact-dependent interaction of CD40-expressing tumor B-cells within the neoplastic follicles or CD40-expressing Reed-Sternberg cells in Hodgkin's disease areas (Carbone et al. (1995) Am. J. Pathol. 147:912-922). However, the mechanism of CD40L-mediated CD40 signaling leading to survival versus cell death responses of malignant B-cells is not completely known. For example, in follicular lymphoma cells, down-regulation of apoptosis-inducing TRAIL molecule (APO-2L) (Ribeiro et al. (1998) British J. Haematol. 103:684-689) and overexpression of Bcl-2, and in the case of B-CLL, down-regulation of CD95 (Fas/APO-1) (Laytragoon-Lewin et al. (1998) Eur. J. Haematol. 61:266-271) have been proposed as mechanisms of survival. In contrast, evidence in follicular lymphoma indicates that CD40 activation leads to up-regulation of TNF (Worm et al. (1994) International Immunol. 6:1883-1890) and CD95 molecules (Plumas et al. (1998) Blood 91:2875-2885).
Given the important role of CD40 signaling in survival and proliferation of neoplastic cells, methods for identifying individuals who would be responsive to treatment regimens that target CD40-expressing tumors and more specifically CD40 signaling are needed.